In principle, the optical resolution of a light microscope, such as a LSM, is limited with regard to diffraction by the laws of physics. For an optimal resolution within these limits particularly illumination configurations are known, such as 4Pi arrangements or arrangements with fields of stationary waves. Here, the resolution can be considerably improved, particularly in the axial direction, in reference to classic LSM.
Using non-linear depopulation processes, the resolution can be further increased in reference to a diffraction-limited confocal LSM. Such a method is described, e.g., in U.S. Pat. No. 5,866,911. Several approaches are known for the depopulation process, such as described in DE 4416558 C2, U.S. Pat. No. 6,633,432, or DE 10325460 A1.
In recent years, various methods to overcome the diffraction limit have been developed and applied in fluorescence microscopy (PALM structured illumination WO 2006127692; EP 1157297 B1).
A presently particularly advantageous method developed for high-resolution fluorescence microscopy is based on the highly-precise localization of individual molecules. It is known that the localization, thus the determination of the position of an individual fluorescent molecule, is not subject to limits of diffraction.
This localization can occur with highly sensitive cameras in the wide field with a precision up to the nm-range, when sufficient protons of the molecule can be detected. In high-resolution microscopy based on localization an image is composed from the molecule positions obtained in this manner. Here it is critical that at any given time only one subset of molecules of the sample are in a fluorescent state so that on average the “closest neighbor” distance of the active molecules is always greater than the PSF (point spread function) of the microscope. This is achieved by using optically or chemically switched fluorophores: in a tightly marked area of a sample, by way of irradiation of suitable conversion wavelength, stochastic subsets of fluorophores are switched in the examined area into the fluorescent state. Here, the density of the spot is adjusted such that a continuous localization of the positions of the molecules is permitted. This optic switching method is used, for example in PhotoActivated Localization Microscopy (PALM). This fundamental method is described in the literature (listed hereinbelow) in detail, using different variations, see literature items [1-6].
Here, the high-resolution methods (PALM, STORM, D-STORM etc.) primarily differ in the selection of the fluorophores and the type of optic switching process.
However, all methods have in common that the localization of the molecules occurs by an imaging process on a highly sensitive camera (e.g., EMCCD). The quasi-punctual light source (molecule) is here displayed by the point spread function (PSF) of the microscope over several camera pixels. The precise position of the molecule in the x/y-level can now be determined either by fitting the known PSF (Gauss) or by a determination of the focus or by a mixture of both (Gaussian mask) (see the literature, citing different algorithms).
Typical precisions of localization range (depending on the experimental conditions) from 5 to 30 nm; this then also represents approximately the lateral resolution of this method. Practically, this calls on the one hand for molecules not being located too close to each other and on the other hand for an illustration of the examined structures as completely as possible, so that many individual images (typically 10,000-20,000) must be taken of the sample. This leads to a rather extensive imaging period as well as to problems with regard to the adjustment of the switching intensity, particularly when the sample and/or the structures of interest are marked very inhomogeneously: in order to prevent losing any information the switching intensity must always be adjusted to the area of the sample marked most densely.
In general, the above-described method based on localized high resolution is limited to surfaces and/or 2 dimensions, because the localization of the individual colorant molecules are considerably more complex in the third dimension (z-direction). The number of accepting individual molecules for illustrating the structures increases accordingly in the three-dimensional case.
Another problem for the three-dimensional high-resolution display in the depth of a sample lies in the limited penetration depth: as already known from the classical fluorescence microscopy and laser-scanning microscopy, the increasingly dispersed excited radiation in the depth of the sample leads to an increase of the background signals with a simultaneous reduction of the actual wanted signal.
In addition to the undesired photo bleaching of sample sections outside the focused level, it also occurs in the PAL-M method that in the depth of the sample undesired switching of the fluorophores occurs by the activating radiation in the layers not presently measured.
In prior art there is the general need for a high-resolution display of fluorescence in three dimensions with high penetration depth and “sectioning” (thus measuring a layer and here avoiding exciting/bleaching and particularly switching the photo-convertible fluorophores in the layers located above or below) and an increase in recording speed.